Cells can be transplanted from a donor to a patient to produce biologics that may be absent, disrupted, or produced at insufficient levels in the patient. More generally, cells can be transplanted to effect a change to alleviate medical symptoms in a patient. However, the introduction of foreign donor cells into a patient often generates a host immune response that can destroy the transplanted cells. Research has focused on methods to minimize or abolish host immune response, thereby maximizing the effectiveness of the transplanted cells.
Animal models and clinical trials suggest a major obstacle in islet transplantation is a high rate of primary nonfunction and early islet destruction after intraportal islet infusion. Acute blood mediated inflammatory injury is largely responsible for functional stunning or destruction of islets and may amplify later immune reactions. Thus, there is a need in the art to generate an islet encapsulation barrier that is “actively” anti-inflammatory while preserving appropriate permeability characteristics for the exchange of nutrient and waste products, as well as passage through the barrier of useful biologics generated by the encapsulated cell. A particularly useful application of this barrier technology is for islet transplantation.
Whole organ pancreatic allografts using current immunosuppressive protocols have an expected graft survival as high as 86% at one year and 74% at 5 years after transplantation (1). Despite these encouraging results, the risk of major perioperative morbidity, the associated complications of chronic immunosuppression therapy, and the persistent shortage of donor organ tissue remain limitations of this approach. As a consequence, pancreas transplantation continues to have a limited role in the management of diabetes (2, 3).
Barrier strategies have focused on the coating of alginate microbeads with a membrane-mimetic film. Indeed, microcapsules/beads offer several distinct advantages over the use of other barrier devices including, greater surface to volume ratio, ease of implantation, and retrievability by lavage and needle aspiration when implanted into the peritoneal cavity. Nonetheless, the currently favored site for clinical islet transplantation involves infusion into the portal vein, where islets lodge in the terminal portal radicals. Relative advantages of the intrahepatic site in comparison to the peritoneal cavity include a substantially higher oxygen tension, as well as greater efficiency in the delivery of nutrients and removal of wastes. Unfortunately, standard microcapsules cannot be used in this site due to their large size (d ˜500-600 μm) and the increased risk of portal vein thrombosis. These large diameters are required to achieve a high rate of islet encapsulation. As a consequence, use of a conformally coated islet barrier, based upon LbL polymer self-assembly, as a starting point for incorporation of membrane-based immunomodulatory proteins and carbohydrates is advantageous. Of note, the use of a conformal coating (i.e. a thin barrier film coated directly on the islet surface) is also associated with an inherent reduction in the large void volume typical of microcapsules, which favors a more efficient exchange of oxygen and nutrients. Indeed, formation of conformal islet barriers comprised of a single layer of adsorbed and photocrosslinked polyethylene oxide show promise in an intraperitoneal murine model (109-111).
As an alternative approach, islet transplantation offers several important advantages over whole organ transplantation. First, islets can be maintained and manipulated more easily than whole organ grafts and may be harvested from donor grafts that otherwise would not be suitable for whole organ transplantation. Second, islet transplantation, in comparison to whole organ grafting, is associated with a considerable reduction in morbidity and mortality, a decrease in intensive care unit utilization, shorter hospital stays, with the promise of achieving major reductions in overall healthcare costs. Finally, the opportunity to use a cell encapsulating barrier offers the potential to circumvent the vigorous humoral and cellular responses of the host; facilitating the use of xenogeneic islets or insulin producing cell lines, and thereby increase the supply of non-human donor tissue (2-10). Indeed, current cell encapsulating membranes are capable of isolating the cell transplant from the effects of direct cell-cell interactions, as well as large macromolecules (>100 kD), such as antibodies or associated immune complexes. Nonetheless, despite the production of a variety of permselective capsular membranes, including multicomponent polymer blends and phase inversion membranes, none have resulted in a clinically effective device for either allo- or xenogeneic cell transplantation. The barriers of the present invention improve immunoisolation by structurally mimicking the capacity of the cell membrane to both limit non-specific cell-cell interactions and control interfacial transport processes. In addition, incorporation of membrane-based immunomodulatory proteins and carbohydrates further minimizes adverse immune response to the transplanted cells. In this manner, both inflammatory and immunological processes that contribute to graft failure are limited by the presence of a biologically functional barrier. The cell conforming barrier disclosed herein is not limited to only transplanted islet cells, but can be utilized for any to-be-transplanted cell.
The design characteristics of an immunoisolation barrier are dictated by an intent to limit the effects of rejection pathways initiated after the implantation of donor tissue. Notably, while some overlap exists, the immunological pathways responsible for autoimmune destruction of isogeneic islets or rejection of allogeneic islets differ from those primarily operative in the rejection of xenogeneic grafts. In the former two cases, islet damage appears to be mediated by a primary ‘Th1’ immune response in which the dominant effector cell is a cytotoxic CD8+ T cell (11-14). Specifically, host CD8+ T cells are activated by donor MHC-peptide complexes expressed on the surface of graft-derived antigen presenting cells; a process that has been referred to as direct antigen presentation. In contrast, rejection of islet xenografts is characterized by a ‘Th2’ response in which CD4+ helper T cells, but not CD8+ cells, play a major role (15-20). In this pathway, termed indirect antigen presentation, host antigen presenting cells display peptides scavenged from free donor proteins to engage CD4+ T cells, which develop into Th2 cells (21-23). In turn, Th2 cells stimulate the maturation of B cells into plasma cells, which secrete xenoantigen specific antibodies. Immune complexes are generated by the binding of newly formed antibodies to xenoantigens, which may lead to the activation of macrophages (MØ) and recruitment of neutrophils to the islet transplant by activation of the complement cascade or by direct binding of antigen-antibody complexes to leukocyte cell surface Fc receptors (24). Although CD8+ reactivity predominates in allograft immunity and CD4+ reactivity is a primary factor in xenograft immunity, these distinctions are not absolute and both pathways may be active to lesser or greater degrees.
Given this framework, cell isolation strategies that prevent cell-cell contact between donor cells and host immune cells block the direct antigen presentation pathway. While the feasibility of attaining this goal has been demonstrated, the capacity of a barrier to limit indirect antigen presentation by preventing the release of graft protein or peptide antigens, shed from the islet surface or liberated from necrotic or apoptotic cells, is difficult. Moreover, once an immune response is initiated, the selective exclusion of low molecular weight cytokines and free radicals that may be released by immune and inflammatory cells in the region of the graft, while simultaneously permitting the passage of insulin, glucose, or other nutrients, has not been achieved. There is a need in the art, therefore, to minimize an immune response while simultaneously permitting exchange across the barrier of selected substances produced by the transplanted cells. This invention addresses this need by incorporating inflammatory and/or thrombogenic inhibitors in a conformal barrier generated by layer-by-layer (LbL) polymer assembly. Such barriers “actively” limit those immune mediated responses related to indirect antigen presentation, in addition to preventing cell-cell interactions that underlie the initiation of the CD8+-dependent pathway. This is achieved by the incorporation of immunomodulatory proteins and carbohydrates into the encapsulation barrier, which limit the activation of macrophages and T cells, as well as the complement pathway. Such a barrier can also limit later induction of an immune response by abrogating early inflammatory graft injury.
Islets from two to four donor organs are typically required to reverse diabetes in a single patient, placing a significant burden on an already limited donor organ supply (26, 27). Moreover, a requirement for successive islet infusions within the portal bed necessitates re-interventions with increased costs, the attendant risk of periprocedural morbidity, and has been associated with increasing portal vein pressures that may indicate the development of a presinusoidal form of portal hypertension. Primary nonfunction may be the consequence of poor functional quality of the grafted tissue, an inadequate mass of transplanted islets, or lack of vascularization of the graft (28). However, substantial evidence now suggests that exposure to an early, nonimmune inflammatory injury is largely responsible for the observed functional stunning or destruction of islets and may well amplify subsequent immune reactions (29-33). The cell encapsulating barriers of the present, therefore, are useful in preserving islet function by limiting early nonimmune inflammatory injury, thereby reducing requirements for donor islet mass.
Although activation of the graft microenvironment by endotoxin (34, 35) and lipopolysaccharides has been postulated to contribute to induction of a local inflammatory response, an acute blood mediated inflammatory reaction is initiated upon intraportal infusion of islets (36-38). Specifically, in animal models and in recent clinical reports, marked activation of coagulation has been noted after islet infusion, despite the presence of heparin in the infusate, as indicated by increases in thrombin-antithrombin (TAT) complexes, prothrombin activation fragments, and fibrinopeptide A. Indeed, others have also observed overt, as well as subclinical episodes of portal vein thrombosis after islet transplantation (39, 40). Prothrombotic triggers include the expression of tissue factor (TF) either by transplanted islets or by locally injured endothelial cells (37, 38). As a consequence of thrombin generation, activated platelets bind to the islet surface and further amplify the coagulation cascade. Notably, thrombin is a direct mediator of inflammation, acting as a chemoattractant for neutrophils and monocytes and stimulating endothelial cells to express monocyte chemoattractant protein-1 (MCP-1) and other chemokines. Thrombin also induces endothelial cell expression of ICAM-1, VCAM-1, and P-selectin, as well as platelet activating factor, all of which leads to further recruitment of platelets and leukocytes to the graft site (41, 42). Likewise, by-products of the thrombin response, including fibrinogen degradation products and fibrin, also act as chemoattractants and serve to localize this inflammatory response by adhesion-dependent processes. Furthermore, thrombin activated endothelial cells leukocytes express oxygen free radicals, IL-1β, TNF-α, IFN-gamma, and iNOS, which can damage islets, inducing either functional impairment or death (43). Consistent with these observations, immunohistochemical analysis of grafts with primary nonfunction has demonstrated robust macrophage infiltration (29, 44).
Both heparin and thrombomodulin have a pronounced inhibitory effect on thrombotic, inflammatory, and redox related responses. For example, heparin dramatically enhances the ability of heparin cofactor II and antithrombin III to inactivate thrombin. Moreover, heparin inhibits the formation of nitric oxide by binding superoxide dismutase (45) and limits complement mediated effects by inhibiting the formation of C3 convertase and the assembly of C5b-9 (46-48). Perhaps of greater physiologic significance is thrombomodulin (TM), a 60 kD type I transmembrane protein that forms a 1:1 molar complex with thrombin (49-53). In the process, TM switches off all known procoagulant/proinflammatory functions of thrombin, and instead channels the catalytic power of the enzyme into complex anticoagulant/anti-inflammatory activities. Specifically, thrombin bound to TM is incapable of cleaving fibrinogen, nor is it able to activate factor V or platelets (54). It is particularly noteworthy, however, that TM significantly enhances the rate of thrombin inactivation by ATIII (˜8-fold) and dramatically accelerates (˜20,000-fold) the ability of thrombin to activate protein C (APC). Activated protein C together with its cofactor protein S inactivates two coagulation factors, Va and VIIIa, thereby preventing the generation of Xa and thrombin, which are critical for the amplification of both inflammatory and coagulation responses. Apart from thrombin and Xa related processes, APC also inhibits mononuclear phagocyte (MØ) activation and the production of pro-inflammatory cytokines, such as TNF-a, IL-1b, which are known to be cytotoxic to islets (55-58). This inhibitory effect has been observed in response to LPS, IFN-gamma, as well as phorbol myristate acetate. APC also suppresses MØ-dependent proliferative responses of T cells, inhibits mixed lymphocyte responses of human and rat mononuclear cells, and when administered systemically prolongs xenograft survival in a guinea pig to rat cardiac transplant model (58). Furthermore, APC limits neutrophil binding to selectins (59), which indirectly reduces the elaboration of cytokines by endothelial cells. It is notable that a variety of pro-inflammatory cytokines downregulate endothelial cell expression of TM with a concomitant decrease in APC production (60). While APC and heparin have been administered systemically as anti-inflammatory agents, their potent anticoagulant activity limits their effective dose range and, therefore, diminishes their potential therapeutic impact.
In addition to thrombin generation, local release of adenine nucleotides, including ATP and ADP, from activated endothelium and platelets further potentiate proinflammatory and prothrombotic events. Specifically, both ATP and ADP are released into the extracellular environment from activated endothelium and are secreted in high concentrations by platelets following their stimulation with exogenous ADP, collagen, thrombin, or activated complement components (61, 62). These purinergic mediators act as a positive feedback stimulus initiating further recruitment and sequestration of platelets and activating endothelial cells. Of interest, both collagen and thrombin-induced platelet responses are critically dependent on the presence of released ADP, which interacts with purinergic type 2 (P2) receptors as a powerful agonist for platelet adhesion and aggregation. Extracellular ATP also interacts with P2 receptors, including P2X7 receptors that induce pore formation in cell membranes, and promotes IL-1 release from macrophages and endothelial cells. Furthermore, both ATP and ADP activate neutrophils and trigger nitric oxide release from endothelial cells. In summary, elevated concentrations of ATP and ADP predispose to thrombosis and inflammation at the vascular wall interface. An important regulator of these events is CD39, which is an endothelial cell transmembrane protein with both ecto-ATPase and ecto-ADPase activities, which rapidly metabolizes extracellular ATP and ADP to AMP (63-65). By reducing local concentrations of ATP and ADP, CD39 represents a physiologically important antithrombotic/anti-inflammatory regulatory mechanism-blocking platelet aggregation and recruitment in response to a wide range of stimuli, as well as other EC and leukocyte mediated pro-inflammatory events (66, 67). Indeed, in an intriguing report intravenous administration of soluble CD39 has been shown to prolong whole organ xenograft survival and abrogate platelet activation and deposition seen in this setting (68, 69). Of note, endothelial cell CD39 expression is rapidly downregulated by reperfusion injury, oxidant stress, or cytokine-mediated EC activation responses, all of which occur at the time of portal islet infusion (64, 70).
A comparatively new generation of polymeric shell has been recently introduced, based on the “layer-by-layer” (LbL) assembly of oppositely charged polymeric species onto an underlying substrate (71). The attractiveness of this strategy is based on the observation that film architectures and thickness are completely determined by the deposition sequence and that many different materials can be incorporated in individual multilayer films (72-76). Furthermore, since the process only involves adsorption from solution, there are no restrictions with respect to the size or topology of the object to be coated. In this regard, this strategy offers a new approach for the fabrication of thin multicomponent films directly on cell surfaces (77-79). Thus, the surface of the pancreatic islet and many of its attendant properties can be re-engineered. TM, heparin, and CD39, as components of a conformal islet coating, provide a rational strategy for generating an “actively” anti-inflammatory barrier that reduces primary islet non-function. Moreover, by abrogating early inflammatory graft injury, later induction of an immune response is limited with improved long-term graft survival. The LbL assembly strategy can be combined with membrane-mimetic strategies, as disclosed in U.S. patent application Ser. No. 10/343,408 filed Jul. 22, 2003, herein incorporated by reference, to further increase transplant efficacy.